Life's diversity is largely based on the versatility of two polymers: polypeptides (i.e. proteins) and polynucleotides (nucleic acids). Information storage and propagation in biological systems is commonly based on just two types of nucleic acids, DNA and RNA. Nucleic acids in particular display unique properties beyond their ability to encode genetic information, which make them important tools in chemistry, biotechnology, nanotechnology and medicine. Nucleic acids also have enormous potential as therapeutics but suffer from systemic constraints inherent in DNA and RNA chemistry such as poor serum/nuclease stability.
Systematic chemical studies have begun to uncover the critical chemical and physico-chemical parameters that have enabled DNA and RNA to serve as the molecular basis for life's genetic systems. Changes to the chemical structure of nucleic acids, including unnatural nucleobases1-3 have been used to investigate the molecular determinants for information storage. Synthetic exploration of alternative backbone linkages4 and ribofuranose congeners5,6 have been explored and have revealed the profound influence of the chemical makeup of backbone and/or sugar (or equivalent) chemistry on nucleic acid properties, structure and conformation. Crucially, only a small subset of chemistries allows cross-polymer information transfer through efficient pairing with DNA or RNA: a prerequisite for the formation of a synthetic genetic system capable of crosstalk with extant biology. However, cross-hybridization experiments alone cannot conclusively determine the capacity of a given chemistry to serve as a genetic system as hybridization does not necessarily preserve information content.
A more thorough examination of the potential of a potential genetic polymer for information storage, propagation and evolution requires a system of replication. In principle, artificial polymers might be synthesized and replicated chemically but non-enzymatic polymerization is usually inefficient and error-prone7 and consequently unattractive as a generic approach despite significant advances in the polymerization of mononucleotide8 or short oligomer (pentamer) units9 using specialized chemistries. Enzymatic polymerization using DNA or RNA polymerases is potentially powerful but is restricted but the tight substrate specificity of natural polymerases. Despite significant progress in understanding determinants of polymerase substrate specificity10 and the engineering of polymerases with expanded substrate spectra11,12, most unnatural nucleotide analogues have remained inadequate polymerase substrates at full substitution for either synthesis and/or as templates for reverse transcription.
DNA and RNA are not only a repository of genetic information for life. They are also unique polymers with remarkable properties: they associate according to well-defined rules, can be assembled into diverse nanostructures of defined geometry, can be evolved to bind ligands and catalyze chemical reactions and can serve as a supramolecular scaffold to arrange chemical groups in space.
Aptamers are a promising class of biomolecular therapeutics based on structured single-stranded nucleic acids with the potential to rival antibodies in some clinical settings. A broad spectrum of both RNA- and DNA-based aptamers have been described directed against a wide-range of targets and several are currently undergoing in clinical trails underlining their potential. However, reagents based on natural nucleic acids such as RNA or DNA have drawbacks with respect to a number of desirable properties for clinical reagents and therapeutics, such as in vivo stability and/or bioavailability. In principle, aptamers may be stabilized (post-selection) by medicinal chemistry approaches and this approach has been validated by Macugen, the 1st aptamers based drug, which has been approved for the treatment of macular degeneration. However, post-selection modifications can alter and/or weaken aptamer structure and target interactions and may modify aptamer specificity, which is a problem.
A wide range of modified nucleotides has been used in SELEX to create aptamers comprising unnatural chemistries. Some of these modifications confer desirable characteristics on the selected aptamers such as increased nuclease resistance and stability but also have drawbacks such as toxicity and increased non-specific interaction with proteins.
Orthogonality (i.e. a lack of interaction/interference with the cellular machinery) and the resulting lack of toxicity, increased nuclease resistance as well as other potentially desirable properties may in principle arise from the use of more radically engineered nucleic acids. However, their application to the aptamer field requires both the design and synthesis of such nucleic acids as well as the generation of custom-made polymerases for their synthesis, replication and evolution. It is a problem that such reagents and polymerases do not exist in the art.
Many novel nucleic acid structures have been built with a view towards increased orthogonality. The challenge here is to design scaffolds that lead to minimal interaction/interference with the cellular genetic machinery while simultaneously maintaining an ability to communicate with it. Notable achievements include attempts at expanding the genetic alphabet (informational orthogonality) and altering the structure or size of nucleobases (steric orthogonality). However, in each of these cases issues of cellular toxicity and/or informational specificity remain.
A different approach towards chemically orthogonal nucleic acids involves the modification of the backbone but leaves the informational nucleobases intact. Replacement of the canonical ribofuranose with other pentoses (or hexoses and tetroses) can indeed have dramatic effects on helical conformation and duplex stability and formation.